Summary It is now well established that repeated cocaine exposure results in persistent increases in spine density in nucleus accumbens GABAergic medium spiny neurons (MSNs), which account for over 90% of neurons in the accumbens). A critical unanswered question is whether these new spines form functional synapses. Our preliminary data suggest that this may indeed be the case as cocaine self-administration induces lasting increases in systems that influence axon guidance in both the nucleus accumbens and VTA. Thus, following prolonged abstinence after cocaine self-administration there are increases in the Slit-Robo system, which repels axon guidance, and Netrin-DSCAM, which promotes axon extension, in the nucleus accumbens. The fact that the Slit-Robo and Netrin-1-DSCAM systems produce opposing effects on axon extension suggests differential cocaine-induced synaptogenesis in accumbens MSNs, which are divided into two categories: D1- or D2-dopamine receptor expressing. Repeated cocaine produces opposing effects in these classes of neurons such that transmission through D1 dopamine receptor (D1DR)-expressing MSNs is favored. Therefore, our over-arching hypothesis is that cocaine self-administration increases dopamine synaptogenesis in D1DR- containing MSNs (through Netrin-DSCAM), while the formation of new dopamine synapses in MSNs expressing D2 dopamine receptors (D2DRs) is suppressed via Slit-Robo. In Specific Aim 1 we will assess cocaine-induced changes in Robo2 and DSCAM mRNA specifically in dopaminergic neurons in the VTA. Similarly, cocaine-mediated alterations in Slit2 and Netrin-1 mRNA will be assessed selectively in D1DR- and D2DR-expressing MSNs in the nucleus accumbens (core and shell subregions). Accumbens core and shell protein levels also will be assessed for Robo2 and DSCAM in dopaminergic terminals as well as Slit2 and Netrin-1 protein levels in D1DR- and D2DR-expressing MSNs. We also will determine the functional effect of Netrin-1 knockdown in the accumbens, which we expect to reverse the incubation of craving (i.e. the time-dependent increase in cocaine cue-evoked drug seeking). Specific Aim 2 focuses on synapse formation in the nucleus accumbens after cocaine self-administration and an extended period of forced abstinence using the new mGRASP technique. We predict cocaine-induced synapse formation selectively in dopaminergic projections to D1DR-expressing MSNs in the nucleus accumbens.